Recently, the health effects of particulate matter (PM) from the atmosphere are growing as a critical issue in Asia. PM are divided by diameter size—for example, PM10 (diameter<10 μm), PM2.5 (diameter<2.5 μm), and PM0.1 (diameter<0.1 μm). PM10 includes all types of PM. Generally, PM consists of particle carbon cores, ions, metal, and organic compounds (Folinsbee, 1993; Li
Aryl hydrocarbon receptors (AhRs) bind to molecular complexes including Hsp90, XAP2, and p23 in cytosol. The AhR group is a basic helix–loop–helix PER/ARNT/SIM family of transcription factors and is regarded as regulators of cell morphology and homeostasis (Shimizu
Exposure to PM results in systemic immune responses due to increasing proinflammatory cytokines such as interleukin (IL)-1, IL-6, and IL-8 in lung epithelial cells (Hetland
Autophagy is a catabolic process characterized by self-digestion through the degradation of cellular constituents to form nutrients and maintain cellular homeostasis (Azad
Fine Dust (PM10-like) (ERM®-EZ100) was purchased from the European Reference Materials (ERM) (St. Louis, MO, USA). Fine Dust particles are composed of the size range of 10 μm (x ≤10 μm). α-naphthoflavone (α-NF) which is known as an inhibitor of AhR and used for positive control, and oleanolic acid were purchased from Sigma Aldrich (St. Louis, MO, USA).
HPLC-DAD analyses were carried out using an Agilent 1260 Inifinity (Agilent, Santa Clara, CA, USA). HPLC columns were silica-based C18 Agilent Zorbax Elclipse Plus (250 mm×4.6 mm, 5 μm) (Agilent), column temperature of 30°C, injection volume of 20 μL, and wavelength of 203 nm was used.
The mobile phase was acetonitrile, water with 0.1% phosphoric acid under an isocratic system.
Human immortalized keratinocyte cell line (HaCaT) was obtained from Amore Pacific Company (Yongin, Korea). Human dermal fibroblasts cell line (HDF) was purchased from the American Type Culture Collection (ATCC) (VA, USA). Cells were cultured in DMEM (Welgene, Gyeongsan, Korea) medium supplemented with 10% Fetal Bovine serum (FBS), and 1% Penicillin/Streptomycin. Cells were maintained at 37°C in a 5% CO2 incubator.
Cells were incubated at a density of 1×104 cells/well in 96-well plates. After 24 h at 37°C, the media was replaced with PM diluted to the appropriate concentrations for 24 h. Next, the cells were washed with DPBS and EZ-cytox reagent (Daeil Lab Service, Seoul, Korea) was added, and cells were incubated at 37°C for 30 min. The absorbance was measured using a microplate reader (Tecan, Mannedorf, Switzerland) at a wavelength of 450 nm.
HaCaT was pre-treated with different concentrations of LL (20 μg/mL), LL-EA (2.5, 5, 10 μg/mL) or oleanolic acid (2.5, 5, 10 μg/mL) for 6 h, and after that PM10 (50 μg/mL) was treated.
For RT-PCR, 1ul each of cDNA and respective primers were added to HiPi PCR premix (ELPIS BIO, Daejeon, Korea). The synthesized cDNA was amplified with the following primers : β–actin sense
HaCaT cells were exposed to PM10 with or without oleanolic acid for 30 min and changed fresh media for 24 h. HaCaT cultured supernatant transferred to HDF cells for 30 min and changed fresh media for 48 h. The HDF cultured supernatant were used for analyzing secreted MMP-1 level.
The supernatant media and cells were collected after treatment. The test for the MMP-1 or the IL-6 were performed according to the manufacturer’s instructions. The protein content of cell was quantified using a BCA protein Assay Reagent Kit (Thermo Scientific, Waltham, MA, USA) with bovine serum albumin as the standard.
Cells were seeded in six-well plates in DMEM. One day later, after washing in DPBS, the cells were treated with diluted sample in DMEM with FBS 2% for pre-determined amounts of time. Following treatment, the cells were washed in DPBS and lysed in RIPA buffer (Noble Bio, Hwaseong, Korea) containing protease inhibitor cocktail (PIC, Sigma Aldrich) and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Aldrich) for 30 min at 4°C. The lysate was subjected to centrifugation at 13,000 rpm for 20 min and the resulting supernatant was stored on ice for immediate use or –20°C for longer term storage. The protein content of the supernatant was quantified using a BCA Protein Assay Reagent Kit (Thermo Scientific) with bovine serum albumin as the standard. Equal amounts of protein were separated by NuPAGE™ 12% Bis-Tris Gel (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membranes. Antibodies against β-actin (1:20,000, Sigma Aldrich), p62 (1:1,000, Abcam) were used at 4°C for 24 h. Blots were then incubated with Horse-radish peroxidase conjugated anti-mouse (1:20,000, Bio-Rad, Hercules, CA, USA) or anti-rabbit (1:5,000, Bethyl, Montgomery, TX, USA) secondary antibodies as appropriate at 4°C for 2 h. Blots were visualized by adding a chemiluminescent substrate (Thermo Scientific) and imaged with a FlourChemE imager (HNS Bio, Seoul, Korea).
The ratio acetonitrile and water containing phosphoric acid as the mobile phase, column temperature of 30°C, and wavelength of 203 nm. Under the proposed analytical conditions, baseline resolution was obtained for all the analyses. Chromatograms of the standards and sample solutions are shown in Fig. 1.
To determine the cytotoxic effects of LL, LL-EA, and oleanolic acid, HaCaT cells were treated with indicated concentrations of LL for 24 h. As shown in Fig. 2, no cytotoxic effects were observed. To determine whether LL inhibits the PM10-induced activation of AhRs in keratinocytes, we analyzed the CYP1A1 messenger RNA (mRNA) level by RT-PCR. As shown in Fig. 3A, 3D, PM10 increased the CYP1A1 mRNA level, although this increase was reduced by LL (20 μg/mL). Also, α-NF, inhibitor of AhR, decreased the CYP1A1 mRNA level. Separately, to determine whether LL-EA and oleanolic acid inhibit the activation of AhRs in keratinocytes, we analyzed the CYP1A1 mRNA level by RT-PCR. PM10 increased the CYP1A1 mRNA level, which was decreased by LL-EA (2.5 μg/mL, 5 μg/mL, and 10 μg/mL) (Fig. 3B, 3E) and oleanolic acid (2.5 μg/mL, 5 μg/mL, and 10 μg/mL) (Fig. 3C, 3F). These results suggested that LL-EA inhibits the PM10-induced activation of AhRs more effectively at a lower concentration when compared with LL.
To determine the effects of LL-EA and oleanolic acid on TNF-α, we analyzed the TNF-α mRNA level by RT-PCR. As shown in Fig. 4, PM10 increased the TNF-α mRNA level and this increase was thereafter reduced by LL-EA (2.5 μg/mL, 5 μg/mL, and 10 μg/mL) and oleanolic acid (2.5 μg/mL, 5 μg/mL, and 10 μg/mL). To determine the effects of LL-EA and oleanolic acid on IL-6, we analyzed the IL-6 protein level by enzyme-linked immunosorbent assay. The PM10 treatment group showed increased IL-6 content in keratinocytes, which was decreased by LL-EA and oleanolic acid. Similarly, α-NF decreased the IL-6 level (Fig. 5). These results suggested that PM10-induced TNF-α and IL-6 levels could be decreased by LL-EA or oleanolic acid.
We performed an enzyme-linked immunosorbent assay to reveal that the PM10-induced MMP-1 protein level is decreased by oleanolic acid in dermal fibroblasts. As shown in Fig. 6, PM10 treatment supernatant increased MMP-1 content in fibroblasts, which was decreased by oleanolic acid treatment supernatant. Therefore, treatment by oleanolic acid in keratinocytes can inhibit the PM10-induced MMP-1 protein level in dermal fibroblasts.
Previous research showed that PM10 increases LC3-II and p62 proteins via the activation of AhRs in keratinocytes (Jang
In a previous study, we demonstrated that AhRs are activated by PM10 in keratinocytes, which increases the expression of the
TNF-α initiates nuclear factor–kappa B (NF-κB) nuclear translocation by dissociating the inhibitory protein I-kBα from NF-κB (Wong
In a recent study, a keratinocyte–fibroblast integrated culture system, which is a more suitable means by which to analyze the directional effects of skin exposure rather than the cocultured method, was used to analyze the MMP-1 protein level (Fernando
In a previous study, we discerned that LC3-II and p62 proteins, which are essential for the formation of autophagosomes, were increased by PM10 in keratinocytes (Jang
In conclusion, oleanolic acid could inhibit the activation of AhRs and decrease the heightened TNF-α, IL-6, and MMP-1 levels induced by PM10. Also, oleanolic acid could protects autophagosome accumulation induced by PM10. Thus, oleanolic acid can protect the skin from PM10 exposure and reduce skin inflammation and wrinkles.
This study was supported by a grant of the Gyeonggi Technology Development Program funded by Gyeonggi Province, Republic of Korea (Grant NO: D171754).